This application relates to a process for DNA replication, and to the application of this process for a variety of purposes.
Replication of DNA and other nucleic acids is a complex natural phenomenon which occurs within all biological systems. To facilitate the exploitation of the resources represented in the diverse genetic materials of the world""s organisms, however, it is desirable to be able to replicate selected DNA sequences under more controlled conditions, for example to produce increased amounts of one sequence. Such replication of selected DNA sequences is required for a great many applications of potential scientific and industrial significance, and has been accomplished by a variety of techniques. These include cloning of the DNA sequences into plasmids or genes, and replication of the plasmid using the DNA replication mechanisms of a host organism, and amplification techniques such as PCR or ligase amplification. Cloning is capable of replicating complete gene sequences, but requires the introduction of the sequences into a host organism, and the subsequent recovery of the duplicated DNA. PCR and similar amplification techniques offer increased flexibility, including the ability to introduce labels and/or sequence variations into the replicated DNA, and avoid the use of a host organism, but are limited in the length of the sequence which can be replicated. Thus, there remains a need for a methodology which will permit the replication of long DNA molecules, while providing the flexibility associated with PCR amplification. It is an object of the present invention to provide such a methodology.
The present invention provides a method for replicating DNA, and in particular for replicating large segments of DNA. In accordance with the invention, a primer is combined with a target DNA molecule to be replicated. The primer is designed to be at least partially homologous to a known site on the target DNA, and to create a D-loop when hybridized with that site. A replisome is then assembled at the D-loop, and this replisome creates a copy of the DNA, starting at the primer binding site. By utilizing two species of D-loop primers which bind to remote sites on the DNA flanking a region to be replicated, large sections of DNA can be replicated in a manner comparable to PCR.
The replicated DNA can be analyzed to detect variations in the genetic sequence of the target, for linkage mapping and as a source of longer DNA molecules having a desired sequence.